We thank Sergio G&#243;mez-Medina for excellent technical support with mouse experiments. This work was supported by funds from the Leibniz Association and the Leibniz Center of Infection. A.L. is a recipient of a pre-doctoral fellowship from the Leibniz Graduate School.

Animal experiments were conducted according to approved protocols and following the guidelines of the German animal protection law. All staff carrying out animal experiments passed training programs according to category B or C of the Federation of European Laboratory Animal Science Associations.
1. Generation of Recombinant Live Attenuated Influenza Vaccines by Reverse Genetics
NOTE: The detailed protocol for the generation of recombinant influenza viruses by reverse...

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The authors have nothing to disclose.

In this study we describe how reverse genetics and chimeric mouse models can be utilized to elucidate the physiologic and molecular mechanisms of vaccine-induced immunity. Influenza reverse genetics is established in many laboratories and has played a chief role in understanding influenza pathogenesis, replication, and transmission17. A key point in our protocol is rescue of cold-adapted influenza vaccines expressing foreign epitopes. While the strategy of introducing short cDNAs into the stalk of the neuramin...

The generation of immunological memory in the absence of disease is the physiological basis of efficient vaccination1. Recently, systems biology-based approaches have revealed that successful vaccines such as the yellow fever vaccine, induce a strong induction of innate immune responses and activation of several subsets of dendritic cells (DCs), which in turn, lead to multilineage activation of antigen-specific T cells2,3. Since DCs are the only immune cell population with the ability to activate antigen-specific na&#239;ve T cells4, the study of their function during vaccination is critical to understand immune responses to vaccines and to design future strategies against challenging pathogens.
A system allowing tracing of different DCs subsets during immune responses to vaccines would be desirable in order to establish an accurate kinetics of DC migration to lymphoid tissues, and therefore to provide insight into the physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. Reverse genetics-based approaches offer the possibility to generate modified, live-attenuated vaccines that can be used experimentally with this purpose. Since its implementation on influenza research, plasmid-based reverse genetics has been widely employed to generate recombinant influenza strains including LAIVs. Standard protocols to rescue recombinant influenza viruses require multi-transfection of highly transfectable cell lines with ambisense plasmids (producing both positive and negative sense RNA) containing the eight influenza viral segments as well as amplification in a permissive system such as Madin-Darby canine kidney (MDCK) cells and/or chicken embryonated eggs5. However, the application of reverse genetics to generate molecular tools in order to study the immune mechanisms of vaccination remains unexplored.
The generation of new mouse models allowing specific depletion of immune cell subsets, including DCs, has opened new possibilities to understand the basic immune mechanisms underlying vaccine-elicited protection. The comparison between DC subset functions in mice and humans has revealed that, to a great extent, mouse and human DCs are functionally homologous6,7, these findings, strongly suggest that the development of mouse models allowing specific depletion of DCs in the steady state and during inflammatory conditions, may serve to understand the physiology of DC responses in humans. In recent years a number of mouse models have been generated carrying transgenes expressing the simian diphtheria toxin (DT) receptor (DTR) under the control of the promoter region of a gene of interest8,9. Since mouse tissues do not naturally express DTR, these models allow conditional depletion of cell subsets carrying the targeted gene of interest upon mouse inoculation with DT. Thus, our ability to deplete specific DCs and other leukocytes in vivo during physiological processes, has been greatly enhanced by the development of DTR-based ro. However, while these transgenic mouse models have been used extensively to understand the ontogeny of the immune system, their application to vaccine development has been scarcely tested. Here, by combining influenza reverse genetics and DTR-based competitive bone marrow chimeras, we propose a method to study the kinetics of vaccine immunity as well as individual gene function during immune responses to vaccines in vivo. The application of this technology for preclinical evaluation of new vaccines against challenging infectious diseases could help to rationalize vaccine design and to test vaccine candidates in vivo.

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			<td>Dulbecco&#180;s Modified Eagle Medium (DMEM 1X)</td>
			<td>Gibco RL-Life Technologies</td>
			<td>41965-039</td>
			<td></td>
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			<td>Opti MEM</td>
			<td>Gibco RL-Life Technologies</td>
			<td>31985-047</td>
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			<td>Lipofectamine 2000</td>
			<td>Invitrogen-Life Technologies</td>
			<td>11668-027</td>
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			<td>Penicillin-Streptomycin (10.000 U/ml)</td>
			<td>PAA</td>
			<td>p11-010</td>
			<td></td>
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			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
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			<td>Embryonated eggs</td>
			<td>Valo biomedia Gmbh</td>
			<td></td>
			<td></td>
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			<td>PBS (1X)</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;M Nylon Filters</td>
			<td>Greiner-Biorad</td>
			<td>542-070</td>
			<td></td>
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			<td>Red Blood Cell Lysing buffer (RBCL) 10X</td>
			<td>BD Bioscience</td>
			<td>555899</td>
			<td></td>
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		<tr>
			<td>CD16/CD32 Mouse BD Fc Block (2.4G2)</td>
			<td>BD Pharmigen</td>
			<td>553142</td>
			<td></td>
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			<td>APC-Anti-mouse SIINFEKL-H2kb (25 D1.16)</td>
			<td>Biolegend</td>
			<td>141605</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Anti-mouse CD11c (HLA3)</td>
			<td>BD Biosciences</td>
			<td>553802</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor 450-Anti-mouse MHCII (Md/114.15.2)</td>
			<td>eBioscience</td>
			<td>48-5321-82</td>
			<td></td>
		</tr>
		<tr>
			<td>Pe-Cy7-Anti-mouse CD11b (M1/70)</td>
			<td>Biolegend</td>
			<td>101216</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp/Cy5.5-Anti-mouse CD103 (2E7)</td>
			<td>Biolegend</td>
			<td>121416</td>
			<td></td>
		</tr>
		<tr>
			<td>PE-Anti-mouse CD45.1 (A20)</td>
			<td>eBioscience</td>
			<td>12-0453-82</td>
			<td></td>
		</tr>
		<tr>
			<td>V500-Anti-mouse CD45.2 (1O4)</td>
			<td>BD Bioscience</td>
			<td>562130</td>
			<td></td>
		</tr>
		<tr>
			<td>PerCp-eFluor710 -Anti-mouse CD8a (53-6.7)</td>
			<td>eBioscience</td>
			<td>46-0081-80</td>
			<td></td>
		</tr>
		<tr>
			<td>APC-Cy7-Anti-mouse CD3&#949; (145-2611)</td>
			<td>Biolegend</td>
			<td>100325</td>
			<td></td>
		</tr>
		<tr>
			<td>eFluor450-Anti-mouse CD4 (GK 1.5)</td>
			<td>eBioscience</td>
			<td>48-0041-80</td>
			<td></td>
		</tr>
		<tr>
			<td>CFSE Proliferation dye</td>
			<td>eBioscience</td>
			<td>65-0850-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Baytril 2.5%</td>
			<td>Bayer</td>
			<td>65-0850-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Dymethil-Sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Ovalbumin&#160;</td>
			<td>Molecular probes&#160;</td>
			<td>O23020</td>
			<td></td>
		</tr>
		<tr>
			<td>Diphteria Toxin (DT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0564</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-TPCK</td>
			<td>Sigma-Aldrich</td>
			<td>T1426</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACsCanto II Flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo cell analysis software 9.5</td>
			<td>Flowjo inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)&#160;</td>
			<td>Life technologies</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Automatic Cell Counter</td>
			<td>Invitrogen-Life Technologies</td>
			<td>C10227</td>
			<td></td>
		</tr>
	</tbody>
</table>

intranasal administration of recombinant influenza vaccines in chimeric mouse models to study mucosal immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

Generation of recombinant live-attenuated influenza vaccines can be achieved by transfection of plasmids encoding the eight segments of influenza virus under the control of bidirectional promoters5. A cold-adapted influenza vaccine usually contains six segments of a cold-adapted strain as well as the HA and NA of the influenza strain of choice (e.g., H1N1) (Figure 1A). The principle of cold-adaptation is based on virus-restricted replication at 33 &#176;C, the temperature of the upper...

Watch this Scientific Journal Video about Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity at JoVE.com

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

intranasal-administration-recombinant-influenza-vaccines-chimeric

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Immunology and Infection

12.4K Views.  Heinrich Pette Institute, Leibniz Institute for Experimental Virology. There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.